Hybrid tomato &#39;e2134649&#39;

ABSTRACT

The present disclosure relates to a new and distinctive hybrid tomato designated ‘2134649’, to the plants of hybrid tomato ‘E2134649’, to the plant parts of hybrid tomato ‘E2134649’ including the fruit, and to methods for producing a hybrid tomato using ‘E2134649’ as a parent. The disclosure further relates to methods for producing a tomato plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to the methods for producing other tomato lines derived from hybrid tomato ‘E2134649’.

FIELD

The present disclosure relates to the field of plant breeding. Inparticular, the present disclosure relates to a new and distinctivetomato, Lycopersicon esculentum, hybrid designated ‘E2134649’.

BACKGROUND

Cultivated and commercial forms of tomato generally belong to a speciesmost frequently referred to as Lycopersicon esculentum Miller (alsoknown as Solanum lycopersicum) that is grown for its fruit and which iswidely used as a fresh market or processed product. As a crop, tomato isgrown commercially wherever environmental conditions permit theproduction of an economically viable yield. The size of tomato fruitsmay range from small to large and there are cherry, plum, pear,standard, and beefsteak types. Tomatoes may be grouped by the amount oftime it takes for the plants to mature fruit for harvest; in general thecultivars are considered to be early, midseason or late-maturing.Tomatoes can also be grouped by the plant's growth habit, which can bedeterminate or indeterminate. Determinate plants tend to grow theirfoliage first, then set flowers that mature into fruit if pollination issuccessful. All of the fruit tend to ripen on a plant at about the sametime. Indeterminate tomatoes start out by growing some foliage, thencontinue to produce foliage and flowers throughout the growing season.These plants will tend to have tomato fruit in different stages ofmaturity at any given time. More recent developments in tomato breedinghave led to a wider array of fruit color. In addition to the standardred ripe color, tomatoes can be creamy white, lime green, pink, yellow,golden, or orange.

The first largest process market and second largest fresh market fortomatoes in the United States is in California, where processingtomatoes are harvested by machine. The majority of fresh market tomatoesare harvested by hand at vine ripe and mature green stages of ripeness.Fresh market tomatoes are available in the United States year round.Process tomato season in California is from late June to September.Process tomatoes are used in many forms, as canned tomatoes, tomatojuice, tomato sauce, puree, paste and catsup. Over the 500,000 acres oftomatoes that are grown annually in the US, approximately 40% are grownfor fresh market consumption, while the remaining are grown forprocessing.

Lycopersicon is a relatively small genus within the extremely large anddiverse family Solanaceae, which is considered to consist of around 90genera including pepper, tobacco, and eggplant. The genus Lycopersiconhas been divide into two subgenera, the esculentum complex whichcontains those species that can easily be crossed with the commercialtomato and the peruvianum complex which contains those species which arecrossed with considerable difficulty (Stevens, M., and Rick, C. M. 1986.Genetics and Breeding. In: The Tomato Crop. A scientific basis forimprovement, pp. 35-109. Atherton, J., Rudich, G. (eds.). Chapman andHall, New York). Due to its value as a crop, L. esculentum Miller hasbecome widely disseminated all over the world. Even if the preciseorigin of the cultivated tomato is still somewhat unclear, it seems tocome from the Americas, being native to Ecuador, Peru and the GalapagosIslands and initially cultivated by Aztecs and Incas as early as 700 AD.Mexico appears to have been the site of domestication and the source ofthe earliest introduction. It is thought that the cherry tomato, L.esculentum var. cerasiforme, is the direct ancestor of modern cultivatedforms.

Tomato is a simple diploid species with twelve pairs of differentiatedchromosomes. The cultivated tomato is self-fertile and almostexclusively self-pollinating. The tomato flowers are hermaphrodites.Commercial cultivars were initially open-pollinated, but most have nowbeen replaced by better yielding hybrids. Due to its wide disseminationand high value, tomato has been intensively bred.

Tomato is an important and valuable field crop. Thus, there is acontinued need for new tomato varieties. In particular, there is a needfor improved tomato varieties that are stable, high yielding, andagronomically sound.

BRIEF SUMMARY

In order to meet these needs, the present disclosure is directed toimproved hybrid tomatoes. In one embodiment, the present disclosure isdirected to a hybrid tomato, Lycopersicon esculentum, seed designated as‘E2134649’ having ATCC Accession Number X1. In one embodiment, thepresent disclosure is directed to a Lycopersicon esculentum tomato plantand parts isolated therefrom produced by growing ‘E2134649’ tomato seed.In another embodiment, the present disclosure is directed to aLycopersicon esculentum plant and parts isolated therefrom having allthe physiological and morphological characteristics of a Lycopersiconesculentum plant produced by growing ‘E2134649’ tomato seed having ATCCAccession Number X1. In still another embodiment, the present disclosureis directed to an F₁ hybrid Lycopersicon esculentum tomato seed, plantsgrown from the seed, and fruit isolated therefrom having ‘E2134649’ as aparent, where ‘E2134649’ is grown from ‘E2134649’ tomato seed havingATCC Accession Number X1.

Tomato plant parts include tomato leaves, ovules, pollen, seeds, tomatofruits, parts of tomato fruits, flowers, cells, and the like. In oneembodiment, the present disclosure is directed to tomato leaves, ovules,pollen, seeds, tomato fruits, parts of tomato fruits, flowers and/orcells isolated from ‘E2134649’ tomato plants. In certain embodiments,the present disclosure is further directed to pollen or ovules isolatedfrom ‘E2134649’ tomato plants. In another embodiment, the presentdisclosure is further directed to protoplasts produced from ‘E2134649’tomato plants. In another embodiment, the present disclosure is furtherdirected to tissue culture of ‘E2134649’ tomato plants, and to tomatoplants regenerated from the tissue culture, where the plant has all ofthe morphological and physiological characteristics of ‘E2134649’tomato. In certain embodiments, tissue culture of ‘E2134649’ tomatoplants is produced from a plant part selected from leaf, anther, pistil,stem, petiole, root, root tip, fruit, seed, flower, cotyledon,hypocotyl, embryo and meristematic cell.

In another embodiment, the present disclosure is further directed to amethod of selecting tomato plants, by a) growing ‘E2134649’ tomatoplants where the ‘E2134649’ plants are grown from tomato seed havingATCC Accession Number X1 and b) selecting a plant from step a). Inanother embodiment, the present disclosure is further directed to tomatoplants, plant parts and seeds produced by the tomato plants where thetomato plants are isolated by the selection method described using‘E2134649’ tomato plants.

In another embodiment, the present disclosure is further directed to amethod of making tomato seeds by crossing a tomato plant grown from‘E2134649’ tomato seed having ATCC Accession Number X1 with anothertomato plant, and harvesting seed therefrom. In still anotherembodiment, the present disclosure is further directed to tomato plants,tomato parts from the tomato plants, and seeds produced therefrom wherethe tomato plant is grown from seed produced by the method of makingtomato seed described using ‘E2134649’ tomato plants. In someembodiments, the tomato plant grown from tomato seed produced by themethod of making tomato seed using ‘E2134649’ tomato plants is atransgenic tomato plant.

In another embodiment, the present disclosure is further directed to amethod of making hybrid tomato ‘E2134649’ by selecting seeds from thecross of one ‘E2134649’ plant with another ‘E2134649’ plant, a sample of‘E2134649’ tomato seed having been deposited under ATCC Accession NumberX1.

According to the present disclosure, there is provided a hybrid tomatoplant designated as ‘E2134649’. This disclosure thus relates to theseeds of hybrid tomato ‘E2134649’, to the plants of hybrid tomato‘E2134649’, as well as to methods for producing a tomato plant producedby crossing hybrid tomato ‘E2134649’ with itself or another tomatoplant. The present disclosure also relates to methods for producing atomato plant containing in its genetic material one or more transgenesand to the transgenic tomato plants produced by that method. Thisdisclosure also relates to methods for producing other tomato cultivarsor hybrids derived from hybrid tomato ‘E2134649’, and to the tomatocultivars and hybrids derived by the use of those methods. Thisdisclosure further relates to tomato seeds and plants produced bycrossing hybrid tomato ‘E2134649’ with another tomato cultivar.

In another embodiment, the present disclosure is directed to methods forproducing a tomato plant containing in its genetic material one or moretransgenes and to the transgenic tomato plant produced by those methods.In some embodiments, the transgenic tomato plant has essentially all thephysiological and morphological characteristics of hybrid tomato‘E2134649’.

In another embodiment, the present disclosure is directed to single geneconverted plants of hybrid tomato ‘E2134649’. The single transferredgene may preferably be a dominant or recessive allele. Preferably, thesingle transferred gene will confer such trait as sex determination,herbicide resistance, insect resistance, resistance for bacterial,fungal, or viral disease, improved harvest characteristics, enhancednutritional quality, or improved agronomic quality. The single gene maybe a naturally occurring tomato gene or a transgene introduced throughgenetic engineering techniques.

In another embodiment, the present disclosure is directed to methods fordeveloping tomato plants in a tomato plant breeding program using plantbreeding techniques including recurrent selection, backcrossing,pedigree breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection and transformation. Markerloci such as restriction fragment polymorphisms or random amplified DNAhave been published for many years and may be used for selection (See,Pierce et al., HortScience (1990) 25:605-615; Wehner T., CucurbitGenetics Cooperative Report, (1997) 20: 66-88; and Kennard et al.,Theoretical Applied Genetics (1994) 89:217-224). Seeds, tomato plants,and parts thereof produced by such breeding methods are also part of thedisclosure.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following descriptions. All references cited herein arehereby incorporated by reference in their entirety.

DETAILED DESCRIPTION

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The selected germplasm is crossed in order torecombine the desired traits and through selection varieties or parentlines are developed. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm. These important traits may include higher yield, fieldperformance, fruit and agronomic quality such as firmness, color,content in soluble solids, acidity and viscosity, resistance to diseasesand insects, and tolerance to drought and heat. As tomato fruits may besubject to mechanical harvesting for processing purposes, i.e. juice,paste, catsup, etc., uniformity of plant characteristics such asgermination, growth rate, maturity and plant uniformity is alsodesirable.

Choice of breeding or selection methods can depend on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from pollinations, and the number ofhybrid offspring from each successful cross.

Each breeding program may include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for at least three years. The best lines can then becandidates for new commercial cultivars. Those still deficient in a fewtraits may be used as parents to produce new populations for furtherselection. These processes, which lead to the final step of marketingand distribution, may take from eight to twelve years from the time thefirst cross or selection is made.

One goal of tomato breeding is to develop new, unique, and geneticallysuperior tomato inbred lines and hybrids. A breeder can initially selectand cross two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. A plant breeder canthen select which germplasms to advance to the next generation. Thesegermplasms may then be grown under different geographical, climatic, andsoil conditions, and further selections can be made during, and at theend of, the growing season. In the case of hybrid variety development,two parental lines may be crossed to produce F₁ progeny. A single-crosshybrid is produced when two inbred lines are crossed to produce an F₁hybrid. Once the parental lines that give the best hybrid performancehave been identified, the hybrid seed can be reproduced indefinitely aslong as the homogeneity of the inbred parent is maintained.Alternatively, a hybrid tomato plant may also serve as a parent in thedevelopment of another hybrid tomato plant.

The development of commercial tomato varieties thus requires thedevelopment of tomato parental lines, the crossing of these lines, andthe evaluation of the crosses. Various breeding methods may be used todevelop tomato varieties from breeding populations and are describedherein. Breeding programs can be used to combine desirable traits fromtwo or more varieties or various broad-based sources into breeding poolsfrom which lines are developed by selfing and selection of desiredphenotypes. The new lines are crossed with other lines and the hybridsfrom these crosses are evaluated to determine which have commercialpotential.

Accordingly, the present disclosure is directed to new hybrid tomato‘E2134649’. Breeding methods involving ‘E2134649’, as well as methods ofproducing and evaluating plants derived from ‘E2134649’, are furtherdescribed herein.

DEFINITIONS

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided.

Allele: The allele is any of one or more alternative form of a gene, allof which alleles relates to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

Attachment point: The point on the tomato fruit where the fruit isconnected to the tomato plant.

Backcrossing: Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

BRIX: Means a percentage by weight of the fruit of sugar in solutionmeasured using a refractometer, wherein the fruit is cut in half and thejuice within the fruit is squeezed onto a lens. The juice on the lens isthen measured by the refractometer.

Determinate tomato: A variety that comes to fruit all at once, thenstops bearing. Determinate varieties are best suited for commercialgrowing since they can be harvested all at once.

Essentially all the physiological and morphological characteristics: Aplant having essentially all the physiological and morphologicalcharacteristics of another plant means a plant having the physiologicaland morphological characteristics, except for the characteristicsderived from the converted gene, of the other plant.

Flesh color: The color of the tomato flesh that can range fromorange-red to dark red when at ripe stage (harvest maturity).

Fruit: A ripened ovary, together with any other structures that ripenwith the ovary and form a unit.

pH: The pH is a measure of acidity. A pH under 4.35 is desirable toprevent bacterial spoilage of finished products. pH rises as fruitmatures.

Plant part: A plant part means any part of a plant including, forexample, a cell, protoplast, embryo, pollen, ovule, flower, leaf, stem,cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther,shoot tip, shoot, fruit and petiole.

Predicted paste bostwick: The predicted paste bostwick is the flowdistance of tomato paste diluted to 12 degrees brix and heated prior toevaluation. Dilution to 12 degrees brix for bostwick measurement is astandard method used by industry to evaluate product consistency. Thelower the number, the thicker the product and therefore more desirablein consistency oriented products such as catsup. The following formulais usually used to evaluate the predicted paste bostwick: Predictedpaste bostwick=−11.53+(1.64*juice brix)+(0.5*juice bostwick).

Regeneration: Regeneration refers to the development of a plant fromtissue culture.

Relative maturity: Relative maturity is an indication of time until atomato genotype is ready for harvest. A genotype is ready for harvestwhen 90% or more of the tomatoes are ripe.

Semi-erect habit: A semi-erect plant has a combination of lateral andupright branching and has an intermediate-type habit between a prostateplant habit, having laterally growing branching with fruits most of thetime on the ground and an erect plant habit has branching going straightup with fruit being off the ground.

Single gene converted: Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered in addition tothe single gene transferred into the inbred via the backcrossingtechnique or via genetic engineering.

Soluble Solids: Soluble solids refer to the percent of solid materialfound in the fruit tissue, the vast majority of which is sugars. Solublesolids are directly related to finished processed product yield ofpastes and sauces. Soluble solids are estimated with a refractometer,and measured as degrees brix.

Quantitative Trait Loci (QTL): Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Uniform ripening: Refers to a tomato that ripens uniformly, i.e., onethat has no green discoloration on the shoulders. The uniform ripeningis controlled by a single recessive gene.

Vegetative propagation: Means taking part of a plant and allowing thatplant part to form roots where plant part is defined as leaf, pollen,embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil,anther, flower, shoot tip, shoot, stem, fruit and petiole.

Viscosity: The viscosity or consistency of tomato products is affectedby the degree of concentration of the tomato, the amount of and extentof degradation of pectin, the size, shape and quality of the pulp, andprobably to a lesser extent, by the proteins, sugars and other solubleconstituents. The viscosity is measured in Bostwick centimeters by usinginstruments such as a Bostwick Consistometer.

Overview of Hybrid Tomato ‘E2134649’

The present disclosure provides a hybrid tomato ‘E2134649’, which hassuperior characteristics. Hybrid tomato ‘E2134649’ has indeterminategrowth and produces small, yellow fruits. In addition, hybrid tomato‘E2134649’ is suitable for greenhouse cultivation.

Additionally, hybrid tomato ‘E2134649’ has shown uniformity andstability for the traits, within the limits of environmental influencefor the traits. The hybrid has been increased with continued observationfor uniformity. No variant traits have been observed or are expected inhybrid tomato ‘E2134649’.

‘E2134649’ has the following morphologic and other characteristics asoutlined in Table 1.

TABLE 1 Variety Description Information For ‘E2134649’ PLANT: Growthtype: Indeterminate Plant height Medium Time of maturity: Medium Type ofculture: Under glass Main use: Fresh market or garden LEAF: Division ofblade: Bipinnate Intensity of green color: Dark PEDUNCLE: Abscissionlayer: Present FRUIT: Size: Small, about 50 g Shape in longitudinalsection: Slightly flattened Ribbing at peduncle end: Absent or very weakNumber of locules: Two or three Green shoulder (before maturity): AbsentColor at maturity: Yellow Firmness: Medium Fruit shelf-life: Short,about 16 days DISEASE AND PEST RESISTANCE: Sensitivity to silvering:Sensitive (susceptible) Meloidogyne incognita (root-knot nematode):Resistant Verticillium dahliae race 0: Susceptible Fusarium oxysporum f.sp. lycopersici race 0: Resistant Fusarium oxysporum f. sp. lycopersicirace 1: Resistant Fusarium oxysporum f. sp. lycopersici race 2:Susceptible Fusarium oxysporium f. sp. radicis lycopersici ResistantCladosporium falvum (Ff) group A Resistant Cladosporium falvum (Ff)group B Resistant Cladosporium falvum (Ff) group C ResistantCladosporium falvum (Ff) group D Resistant Cladosporium falvum (Ff)group E Resistant Tomato Mosaic Virus (ToMV) strain 0 Resistant TomatoMosaic Virus (ToMV) strain 1 Resistant Tomato Mosaic Virus (ToMV) strain2 Resistant

Hybrid tomato ‘E2134649’ is similar to tomato ‘Adoration’. While similarto tomato ‘Adoration’, there are differences as shown in Table 2. Column1 of Table 2 shows the plant characteristics being compared, column 2shows the characteristics of hybrid tomato ‘E2134649’, and column 3shows the characteristics of tomato ‘Adoration’.

TABLE 2 Comparison of Characteristics Between ‘E2134649’ and ‘Adoration’Characteristic ‘E2134649’ ‘Adoration’ Fruit color Yellow Red Plantstrength Medium Strong Leaf length Medium Long Green back Absent Present

Further Embodiments

This present disclosure is further directed to methods for producing atomato plant by crossing a first parent tomato plant with a secondparent tomato plant where either the first or second parent tomato plantis hybrid tomato ‘E2134649’. Further, both first and second parenttomato plants can come from hybrid tomato ‘E2134649’. All plantsproduced using hybrid tomato ‘E2134649’ as a parent are within the scopeof the disclosure, including plants derived from hybrid tomato‘E2134649’.

Further, the disclosure is directed to methods for producing a‘E2134649’-derived tomato plant by crossing hybrid tomato ‘E2134649’with a second tomato plant and growing the progeny seed, and repeatingthe crossing and growing steps with the ‘E2134649’-derived plant from 0to 7 times. Thus, any such methods using hybrid tomato ‘E2134649’ areincluded in this disclosure: selfing, backcrosses, hybrid production,crosses to populations, and the like. Plants produced using hybridtomato ‘E2134649’ as a parent are presented herein, including plantsderived from ‘E2134649’. Advantageously, ‘E2134649’ may be used incrosses with other tomato plants including, for example, other tomatohybrids, to produce first generation (F₁) tomato hybrid seeds and plantswith superior characteristics.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which tomato plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, leaves,stems, and the like.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering plant genomes to contain and express foreigngenes, or additional, or modified versions of native, or endogenous,genes (perhaps driven by different promoters) in order to alter thetraits of a plant in a specific manner. Such foreign additional and/ormodified genes are referred to herein collectively as “transgenes.”Several methods for producing transgenic plants have been developed, andthe present disclosure, in particular embodiments, also relates totransformed versions of plants. In particular, the present disclosurerelates to transformed versions of hybrid tomato ‘E2134649’.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector contains DNA includinga gene under control of or operatively linked to a regulatory element(for example, a promoter). The expression vector may contain one or moresuch operably linked gene/regulatory element combinations. The vector(s)may be in the form of a plasmid, and can be used alone or in combinationwith other plasmids, to provide transformed tomato plants usingtransformation methods as described herein to incorporate transgenesinto the genetic material of the tomato plant(s).

Expression Vectors for Tomato Transformation

Marker Genes

Expression vectors include at least one genetic marker operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. Positive selection methods arealso known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalswhich confers resistance to kanamycin (Fraley et al., Proc. Natl. Acad.Sci. U.S.A., 80:4803 (1983)). Another commonly used selectable markergene is the hygromycin phosphotransferase gene which confers resistanceto the antibiotic hygromycin (Vanden Elzen et al., Plant Mol. Biol.,5:299 (1985)).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant (Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol.7:171 (1986)). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil (Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988)).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643(1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include alpha-glucuronidase (GUS),alpha-galactosidase, luciferase and chloramphenicol, acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al.,EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available (Molecular Probes publication2908, IMAGENE GREEN, p. 1-4 (1993) and Naleway et al., J. Cell Biol.115:151 a (1991)). More recently, a gene encoding Green FluorescentProtein (GFP) has been utilized as a marker for gene expression inprokaryotic and eukaryotic cells (Chalfie et al., Science 263:802(1994)). GFP and mutants of GFP may be used as screenable markers.

Promoters

Genes included in expression vectors may be driven by a nucleotidesequence containing a regulatory element, for example, a promoter.Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control include,for example, promoters that preferentially initiate transcription incertain tissues such as leaves, roots, seeds, fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as“tissue-preferred.” Promoters that initiate transcription only in acertain tissue are referred to as “tissue-specific.” A “cell-type”specific promoter primarily drives expression in certain cell types inone or more organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may affect transcription byinducible promoters include, for example, anaerobic conditions or thepresence of light. Tissue-specific, tissue-preferred, cell typespecific, and inducible promoters constitute the class of“non-constitutive” promoters. A “constitutive” promoter is a promoterthat is active under most environmental conditions.

Inducible Promoters: An inducible promoter is operably linked to a genefor expression in tomato. Optionally, the inducible promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in tomato. Inducible promotersmay regulate transcription in response to an inducing agent.

Any inducible promoter can be used herein. See Ward et al., Plant Mol.Biol. 22:361-366 (1993). Exemplary inducible promoters may include, forexample, that from the ACEI system which responds to copper (Meft etal., Proc. Natl. Acad. Sci. U.S.A. 90:4567-4571 (1993)); In2 gene frommaize which responds to benzenesulfonamide herbicide safeners (Hersheyet al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen.Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol.Gen. Genetics 227:229-237 (1991). A particularly preferred induciblepromoter is a promoter that responds to an inducing agent to whichplants do not normally respond. An exemplary inducible promoter is theinducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

Constitutive Promoters: A constitutive promoter is operably linked to agene for expression in tomato or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in tomato.

Many different constitutive promoters can be utilized herein. Exemplaryconstitutive promoters may include, for example, the promoters fromplant viruses such as the 35S promoter from CaMV (Odell et al., Nature313:810-812 (1985) and the promoters from such genes as rice actin(McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (Christensen etal., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., PlantMol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet.81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984)) andmaize H3 histone (Lepetit et al., Mol. Gen. Genetics 231:276-285 (1992)and Atanassova et al., Plant Journal 2 (3): 291-300 (1992)). The ALSpromoter, Xbal/Ncol fragment 5′ to the Brassica napus ALS3 structuralgene (or a nucleotide sequence similarity to said Xbal/Ncol fragment),represents a particularly useful constitutive promoter. See PCTapplication WO 96/30530.

Tissue-specific or Tissue-preferred Promoters: A tissue-specificpromoter is operably linked to a gene for expression in tomato.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in tomato. Plants transformed with a transgeneoperably linked to a tissue-specific promoter produce the transgenicprotein product exclusively, or preferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized herein.Exemplary tissue-specific or tissue-preferred promoters may include, forexample, a root-preferred promoter, such as that from the phaseolin gene(Murai et al., Science 23:476-482 (1983) and Sengupta-Gopalan et al.,Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324 (1985)); a leaf-specific andlight-induced promoter such as that from cab or rubisco (Simpson et al.,EMBO J. 4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582(1985)); an anther-specific promoter such as that from LAT52 (Twell etal., Mol. Gen. Genetics 217:240-245 (1989)); a pollen-specific promotersuch as that from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168(1993)) or a microspore-preferred promoter such as that from apg (Twellet al., Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of a protein produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall or mitochondrion or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine during protein synthesis andprocessing where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderonet al., Cell 39:499-509 (1984), Steifel et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present disclosure, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein can then beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a tomato plant. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology, CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful, for example, in geneticcomparisons where the genetic maps of two plants are compared. Wang etal. discuss “Large Scale Identification, Mapping and Genotyping ofSingle-Nucleotide Polymorphisms in the Human Genome,” Science,280:1077-1082, 1998, and similar capabilities are becoming increasinglyavailable for the tomato genome. Map comparisons may involve, forexample, hybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques. SNPs may also be used alone or in combinationwith other techniques.

Likewise, by means of the present disclosure, plants can be geneticallyengineered to express various phenotypes of horticultural interest.Through the transformation of tomato the expression of genes can bealtered to enhance disease resistance, insect resistance, herbicideresistance, horticultural quality and other traits. Transformation canalso be used to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to tomato as well as non-native DNAsequences can be transformed into tomato and used to alter levels ofnative or non-native proteins. Various promoters, targeting sequences,enhancing sequences, and other DNA sequences can be inserted into thegenome for the purpose of altering the expression of proteins. Reductionof the activity of specific genes (also known as gene silencing, or genesuppression) is desirable for several aspects of genetic engineering inplants.

Many techniques for gene silencing are well known to one of skill in theart, including, for example, knock-outs (such as by insertion of atransposable element such as mu (Vicki Chandler, The Maize Handbook ch.118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Loxor other site specific integration site, antisense technology (see,e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453,566; and 5,759,829); co-suppression (e.g., Taylor(1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech.8(12):340-344; Flavell (1994) PNAS USA 91:3490-3496; Finnegan et al.(1994) Bio/Technology 12: 883-888; and Neuhuber et al. (1994) Mol. Gen.Genet. 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141;Zamore et al. (2000) Cell 101:25-33; and Montgomery et al. (1998) PNASUSA 95:15502-15507), virus-induced gene silencing (Burton, et al. (2000)Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant Bio.2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature334: 585-591); hairpin structures (Smith et al. (2000) Nature407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman & Sakai(2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al. (1992) EMBOJ. 11:1525; and Perriman et al. (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO03/048345; and WO 00/42219); and other methods or combinations of theabove methods known to those of skill in the art.

Likewise, by means of the present disclosure, other genes can beexpressed in transformed plants, such as transformed versions of hybridtomato ‘E2134649’. More particularly, plants can be geneticallyengineered to express various phenotypes of interest. Exemplary genesimplicated in this regard may include, for example, those categorizedbelow.

Genes that Confer Resistance to Pests or Disease

Plant disease resistance genes: Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety, such as a tomato variety, can betransformed with one or more cloned resistance genes to engineer plantsthat are resistant to specific pathogen strains. See, for example, Joneset al., Science 266:789 (1994) (cloning of the tomato Cf-9 gene forresistance to Cladosporium falvum); Martin et al., Science 262:1432(1993) (tomato Pto gene for resistance to Pseudomonas syringae pv.tomato encodes a protein kinase); Mindrinos et al. Cell 78:1089 (1994)(Arabidopsis RSP2 gene for resistance to Pseudomonas syringae), McDowell& Woffenden, (2003) Trends Biotechnol. 21(4): 178-83 and Toyoda et al.,(2002) Transgenic Res. 11 (6):567-82.

A gene conferring resistance to a pest, such as a nematode: See, forexample, PCT Application WO 96/30517; PCT Application WO 93/19181.

A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon: See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

A lectin: See, for example, Van Damme et al., Plant Molec. Biol. 24:25(1994), who disclose the nucleotide sequences of several Clivia miniatamannose-binding lectin genes.

A vitamin-binding protein, such as avidin: See, for example, PCTapplication U.S. Ser. No. 93/06487 which teaches the use of avidin andavidin homologues as larvicides against insect pests.

An enzyme inhibitor, for example, a protease or proteinase inhibitor oran amylase inhibitor: See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof: See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest: For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata); Chattopadhyay et al. (2004) CriticalReviews in Microbiology 30 (1): 33-54 2004; Zjawiony (2004) J Nat Prod67 (2): 300-310; Carlini & Grossi-de-Sa (2002) Toxicon, 40 (11):1515-1539; Ussuf et al. (2001) Curr Sci. 80 (7): 847-853; andVasconcelos & Oliveira (2004) Toxicon 44 (4): 385-403. See also U.S.Pat. No. 5,266,317 to Tomalski et al., which discloses genes encodinginsect-specific, paralytic neurotoxins.

An insect-specific venom produced in nature by a snake, a wasp, etc. Forexample, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

An enzyme responsible for hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

An enzyme involved in the modification, including the post-translationalmodification, of a biologically active molecule; for example, aglycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease,a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, akinase, a phosphorylase, a polymerase, an elastase, a chitinase and aglucanase, whether natural or synthetic: See, for example, PCTapplication WO 93/02197 (Scott et al.), which discloses the nucleotidesequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained from the ATCC underAccession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem.Molec. Biol. 23:691 (1993), who teach the nucleotide sequence of a cDNAencoding tobacco hornworm chitinase, and Kawalleck et al., Plant Molec.Biol. 21:673 (1993), who provide the nucleotide sequence of the parsleyubi4-2 polyubiquitin gene, U.S. Pat. Nos. 7,145,060, 7,087,810 and6,563,020.

A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

A hydrophobic moment peptide: See, for example, PCT application WO95/16776 and U.S. Pat. No. 5,580,852, which disclose peptide derivativesof tachyplesin which inhibit fungal plant pathogens, and PCT applicationWO 95/18855 and U.S. Pat. No. 5,607,914 which teaches syntheticantimicrobial peptides that confer disease resistance.

A membrane permease, a channel former or a channel blocker. For example,see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virusand tobacco mosaic virus.

An insect-specific antibody or an immunotoxin derived therefrom. Thus,an antibody targeted to a critical metabolic function in the insect gutwould inactivate an affected enzyme, killing the insect. See, forexample, Taylor et al., Abstract #497, Seventh Int'l Symposium onMolecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

A virus-specific antibody: See, for example, Tavladoraki et al., Nature366:469 (1993), who show that transgenic plants expressing recombinantantibody genes are protected from virus attack.

A developmental-arrestive protein produced in nature by a pathogen or aparasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitate fungalcolonization and plant nutrient release by solubilizing plant cell wallhomo-α-1,4-D-galacturonase. See, for example, Lamb et al.,Bio/Technology 10:1436 (1992). The cloning and characterization of agene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2:367 (1992).

A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

Genes involved in the Systemic Acquired Resistance (SAR) Response and/orthe pathogenesis-related genes. For example, see Briggs, S., CurrentBiology, 5(2) (1995); Pieterse & Van Loon (2004) Curr. Opin. Plant Bio.7(4):456-64 and Somssich (2003) Cell 113(7):815-6.

Antifungal genes: See Cornelissen and Melchers, Plant Physiol.,101:709-712 (1993); Parijs et al., Planta 183:258-264 (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998). Also seeU.S. Pat. No. 6,875,907.

Detoxification genes, such as for fumonisin, beauvericin, moniliforminand zearalenone and their structurally related derivatives. For example,see U.S. Pat. No. 5,792,931.

Cystatin and cysteine proteinase inhibitors: See, for example, U.S. Pat.No. 7,205,453.

Defensin genes: See, for example, WO 03/000863 and U.S. Pat. No.6,911,577.

Genes conferring resistance to nematodes: See, for example, PCTApplication WO 96/30517; PCT Application WO 93/19181, WO 03/033651 andUrwin et al., Planta 204:472-479 (1998), Williamson (1999) Curr OpinPlant Bio. 2(4):327-31.

Genes that confer resistance to Phytophthora root rot, such as the Rps1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps 3-a,Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes. See,for example, Shoemaker et al., Phytophthora Root Rot Resistance GeneMapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

Genes that confer resistance to Brown Stem Rot: See, for example, thosedescribed in U.S. Pat. No. 5,689,035.

Genes that Confer Resistance to an Herbicide

An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea: See, for example, exemplary genes inthis category that code for mutant ALS and AHAS enzyme as described byLee et al., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet.80:449 (1990), respectively.

Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), and pyridinoxy or phenoxy proprionic acidsand cyclohexanediones (ACCase inhibitor-encoding genes): See, forexample, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses thenucleotide sequence of a form of EPSPS which can confer glyphosateresistance. U.S. Pat. No. 5,627,061 to Barry et al. also describes genesencoding EPSPS enzymes. See also U.S. Pat. Nos. 6,566,587; 6,338,961;6,248,876 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908;5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366;5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE37,287 E; and U.S. Pat. No. 5,491,288; and international publicationsEP1173580; WO 01/66704; EP1173581 and EP1173582. Glyphosate resistanceis also imparted to plants that express a gene that encodes a glyphosateoxidoreductase enzyme as described more fully in U.S. Pat. Nos.5,776,760 and 5,463,175. In addition glyphosate resistance can beimparted to plants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. application Ser. No.10/427,692. A DNA molecule encoding a mutant aroA gene can be obtainedunder ATCC accession number 39256, and the nucleotide sequence of themutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Europeanpatent application No. 0 333 033 to Kumada et al., and U.S. Pat. No.4,975,374 to Goodman et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a PAT gene is provided inEuropean application No. 0 242 246 to Leemans et al. DeGreef et al.,Bio/Technology 7:61 (1989) describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2, and Acc2-S3 genes described byMarshall et al., Theor. Appl Genet. 83:435 (1992).

An herbicide that inhibits photosynthesis, such as a triazine (psbA andgs+ genes) and a benzonitrile (nitrilase gene): See, for example,Przibila et al., Plant Cell 3:169 (1991), describe the transformation ofChlamydomonas with plasmids encoding mutant psbA genes. Nucleotidesequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648to Stalker and DNA molecules containing these genes are available underATCC Accession Nos. 53435, 67441 and 67442. Cloning and expression ofDNA coding for a glutathione S-transferase is described by Hayes et al.,Biochem. J. 285:173 (1992).

Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See, for example, Hattori et al.,Mol. Gen. Genet. 246:419, 1995. Other genes that confer tolerance toherbicides include a gene encoding a chimeric protein of rat cytochromeP4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al.,Plant Physiol., 106:17, 1994), genes for glutathione reductase andsuperoxide dismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995),and genes for various phosphotransferases (Datta et al., Plant Mol.Biol. 20:619, 1992).

Protoporphyrinogen oxidase (protox) is necessary for the production ofchlorophyll. The protox enzyme serves as the target for a variety ofherbicidal compounds. These herbicides also inhibit growth of differentspecies of plants present. The development of plants containing alteredprotox activity which are resistant to these herbicides are describedin, for example, U.S. Pat. Nos. 6,288,306; 6,282,837; 5,767,373; andinternational publication WO 01/12825.

Genes that Confer or Contribute to a Value-Added Trait

Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See, for example, Knultzon et al., Proc.Natl. Acad. Sci. USA 89:2625 (1992).

Decreased phytate content: 1) Introduction of a phytase-encoding geneenhances breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) Up-regulation of a gene that reducesphytate content. This, for example, could be accomplished by cloning andthen re-introducing DNA associated with one or more of the alleles, suchas the LPA alleles, identified in maize mutants characterized by lowlevels of phytic acid, such as in Raboy et al., Maydica 35: 383 (1990)and/or by altering inositol kinase activity as in WO 02/059324,US2003/0009011, WO 03/027243, US2003/0079247, WO 99/05298, U.S. Pat. No.6,197,561, U.S. Pat. No. 6,291,224, U.S. Pat. No. 6,391,348, WO2002/059324, U.S. Pat. No. 2003/0079247, WO98/45448, WO99/55882,WO01/04147.

Impacting carbohydrate compositions by, for example, transforming plantswith a gene coding for an enzyme that alters the branching pattern ofstarch, or a gene altering thioredoxin such as NTR and/or TRX (See U.S.Pat. No. 6,531,648) and/or a gamma zein knock out or mutant such as cs27or TUSC27 or en27 (See U.S. Pat. No. 6,858,778 and US2005/0160488,US2005/0204418). See Shiroza et al., J. Bacteriol. 170: 810 (1988)(nucleotide sequence of Streptococcus mutans fructosyltransferase gene),Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotide sequenceof Bacillus subtilis levansucrase gene), Pen et al., Bio/Technology 10:292 (1992) (production of transgenic plants that express Bacilluslicheniformis alpha-amylase), Elliot et al., Plant Molec. Biol. 21: 515(1993) (nucleotide sequences of tomato invertase genes), Sogaard et al.,J. Biol. Chem. 268: 22480 (1993) (site-directed mutagenesis of barleyalpha-amylase gene), and Fisher et al., Plant Physiol. 102: 1045 (1993)(maize endosperm starch branching enzyme II), WO 99/10498 (improveddigestibility and/or starch extraction through modification ofUDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref 1, HCHL, C4H), U.S. Pat.No. 6,232,529 (method of producing high oil seed by modification ofstarch levels (AGP)). The fatty acid modification genes mentioned abovemay also be used to affect starch content and/or composition through theinterrelationship of the starch and oil pathways.

Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols: For example, see U.S. Pat. Nos. 6,787,683and 7,154,029 and WO 00/68393 involving the manipulation of antioxidantlevels through alteration of a phytl prenyl transferase (ppt), WO03/082899 through alteration of a homogentisate geranyl transferase(hggt).

Genes that Control Male Sterility

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on,”the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT: See, for example, international publication WO 01/29237.

Introduction of various stamen-specific promoters: See, for example,international publications WO 92/13956 and WO 92/13957.

Introduction of the barnase and the barstar genes: See, for example,Paul et al., Plant Mol. Biol. 19:611-622, 1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. No. 5,859,341; U.S. Pat. No. 6,297,426; U.S.Pat. No. 5,478,369; U.S. Pat. No. 5,824,524; U.S. Pat. No. 5,850,014;and U.S. Pat. No. 6,265,640.

Genes that Create a Site for Site Specific DNA Integration

Introduction of FRT sites that may be used in the FLP/FRT system and/orLox sites that may be used in the Cre/Loxp system for site-specific DNAintegration. For example, see Lyznik, et al., Site-SpecificRecombination for Genetic Engineering in Plants, Plant Cell Rep (2003)21:925-932 and WO 99/25821. Other systems that may be used include theGin recombinase of phage Mu (Maeser et al., 1991; Vicki Chandler, TheMaize Handbook ch. 118 (Springer-Verlag 1994), the Pin recombinase of E.coli (Enomoto et al., 1983), and the R/RS system of the pSR1 plasmid(Araki et al., 1992).

Genes that Affect Abiotic Stress Resistance

Genes that affect abiotic stress resistance (including, for example,flowering and fruit development, drought resistance or tolerance, coldresistance or tolerance, and salt resistance or tolerance) and increasedyield under stress: For example, see: WO 00/73475 where water useefficiency is altered through alteration of malate; U.S. Pat. No.5,892,009, U.S. Pat. No. 5,965,705, U.S. Pat. No. 5,929,305, U.S. Pat.No. 5,891,859, U.S. Pat. No. 6,417,428, U.S. Pat. No. 6,664,446, U.S.Pat. No. 6,706,866, U.S. Pat. No. 6,717,034, U.S. Pat. No. 6,801,104, WO2000/060089, WO 2001/026459, WO 2001/035725, WO 2001/034726, WO2001/035727, WO 2001/036444, WO 2001/036597, WO 2001/036598, WO2002/015675, WO 2002/017430, WO 2002/077185, WO 2002/079403, WO2003/013227, WO 2003/013228, WO 2003/014327, WO 2004/031349, WO2004/076638, WO 98/09521, and WO 99/38977 describing genes, includingCBF genes and transcription factors effective in mitigating the negativeeffects of freezing, high salinity, and drought on plants, as well asconferring other desirable traits; US 2004/0148654 and WO 01/36596 whereabscisic acid is altered in plants resulting in improved plant phenotypesuch as increased yield and/or increased tolerance to abiotic stress; WO2000/006341, WO 04/090143, U.S. application Ser. No. 10/817,483 and U.S.Pat. No. 6,992,237 where cytokinin expression is modified resulting inplants with increased stress tolerance, such as drought tolerance,and/or increased yield. Also see WO 02/02776, WO 2003/052063,JP2002281975, U.S. Pat. No. 6,084,153, WO 01/64898, U.S. Pat. Nos.6,177,275 and 6,107,547 (enhancement of nitrogen utilization and alterednitrogen responsiveness). For ethylene alteration, see US 20040128719,US 20030166197 and WO 2000/32761. For plant transcription factors ortranscriptional regulators of abiotic stress, see e.g. US 20040098764 orUS 20040078852.

Other genes and transcription factors that affect plant growth and othertraits, such as yield, flowering, plant growth and/or plant structure,can be introduced or introgressed into plants. See, for example, WO97/49811 (LHY), WO 98/56918 (ESD4), WO 97/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 96/14414 (CON), WO96/38560, WO 01/21822 (VRN1), WO 00/44918 (VRN2), WO 99/49064 (GI), WO00/46358 (FR1), WO 97/29123, U.S. Pat. No. 6,794,560, U.S. Pat. No.6,307,126 (GAI), WO 99/09174 (D8 and Rht), and WO 2004/076638 and WO2004/031349 (transcription factors).

Methods for Tomato Transformation

Numerous methods for plant transformation have been developed includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc. Boca Raton, 1993) pages67-88. In addition, expression vectors and in-vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

Agrobacterium-mediated Transformation: One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

Direct Gene Transfer: Alternatives to Agrobacterium-mediatedtransformation exist such as, for example, direct gene transfer. Agenerally applicable method of plant transformation ismicroprojectile-mediated transformation where DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987); Sanford, J. C., Trends Biotech. 6:299(1988); Klein et al., Bio/Tech. 6:559-563 (1988); Sanford, J. C. PhysiolPlant 7:206 (1990); Klein et al., Biotechnology 10:268 (1992). See alsoU.S. Pat. No. 5,015,580 (Christou et al.), issued May 14, 1991 and U.S.Pat. No. 5,322,783 (Tomes, et al.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. See, for example, Zhang et al., Bio/Technology 9:996(1991). Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes et al., EMBO J.,4:2731 (1985); Christou et al., Proc Natl. Acad. Sci. USA 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-ornithine has also been reported. Hain etal., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant CellPhysiol. 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues have also been described (Donn et al., In Abstracts of VIIthInternational Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spenceret al., Plant Mol. Biol. 24:51-61 (1994)).

Following transformation of target tomato tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods well known in the art.

The foregoing methods for transformation may be used for producing atransgenic variety are merely exemplary. One of skill in the art mayrecognize additional transformation techniques that may be used toproduce new tomato varieties described herein. A transgenic variety maybe crossed with another (non-transformed or transformed) variety inorder to produce a new transgenic variety. Alternatively, a genetictrait that has been engineered into a particular tomato line could bemoved into another line using traditional backcrossing techniques thatare well known in the plant breeding arts. For example, a backcrossingapproach could be used to move an engineered trait from a public varietyinto a desirable hybrid, or from a variety containing a foreign gene inits genome into a variety or varieties that do not contain that gene. Asused herein, “crossing” can refer to a simple X by Y cross or theprocess of backcrossing, depending on the context.

Genetic Marker Profile Through SSR and First Generation Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety ora related variety or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, and Single Nucleotide Polymorphisms (SNPs). Forexample, see Cregan et. al, “An Integrated Genetic Linkage Map of theSoybean Genome” Crop Science 39:1464-1490 (1999), and Berry et al.,Assessing Probability of Ancestry Using Simple Sequence Repeat Profiles:Applications to Maize Inbred Lines and Soybean Varieties” Genetics165:331-342 (2003).

Particular markers used for these purposes may include any type ofmarker and marker profile which provides a means of distinguishingvarieties. One method of comparison is to use only homozygous loci forhybrid tomato ‘E2134649’.

Primers and PCR protocols for assaying these and other markers aredisclosed in the Soybase (sponsored by the USDA Agricultural ResearchService and Iowa State University). In addition to being used foridentification of hybrid tomato ‘E2134649’ and plant parts and plantcells of hybrid tomato ‘E2134649’, the genetic profile may be used toidentify a tomato plant produced through the use of hybrid tomato‘E2134649’ or to verify a pedigree for progeny plants produced throughthe use of hybrid tomato ‘E2134649’. The genetic marker profile is alsouseful in breeding and developing backcross conversions.

The present disclosure relates to tomato varieties characterized bymolecular and physiological data obtained from the representative sampleof said variety deposited with the American Type Culture Collection(ATCC). Further provided by the disclosure is a tomato plant formed bythe combination of one of the disclosed tomato plants or plant cellswith another tomato plant or cell and containing the homozygous allelesof the variety.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be informative in linkage analysis relative toother marker systems in that multiple alleles may be present. Anotheradvantage of this type of marker is that, through use of flankingprimers, detection of SSRs can be achieved, for example, by thepolymerase chain reaction (PCR). The PCR detection involves the use oftwo oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA, repeated cycles of heat denaturation of the DNA followedby primer annealing to complementary sequences at low temperatures, andextension of the annealed primers with DNA polymerase. Followingamplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment.

The SSR profile of tomato varieties such as hybrid tomato ‘E2134649’ canbe used to identify tomato plants having that tomato variety as aparent, since such progeny tomato plants will contain the samehomozygous alleles as the parent. For tomato varieties that areessentially homozygous at all relevant loci, most loci should have onlyone type of allele present. In contrast, a genetic marker profile of anF₁ progeny should be the sum of those parents, e.g., if one parent washomozygous for allele x at a particular locus, and the other parenthomozygous for allele y at that locus, then the F₁ progeny will be xy(heterozygous) at that locus. Subsequent generations of progeny producedby selection and breeding are expected to be of genotype x (homozygous),y (homozygous), or xy (heterozygous) for that locus position. When theF₁ plant is selfed or sibbed for successive filial generations, thelocus should be either x or y for that position.

In addition, plants and plant parts substantially benefiting from theuse hybrid tomato ‘E2134649’ containing a backcross conversion,transgene, or genetic sterility factor, may be identified by having amolecular marker profile with a high percent identity to hybrid tomato‘E2134649’ used in their development. Such a percent identity might be95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to hybrid tomato‘E2134649’ used to develop the plant and/or plant part.

The SSR profile of hybrid tomato ‘E2134649’ can also be used to identifyessentially derived varieties and other progeny varieties developed fromthe use of hybrid tomato ‘E2134649’, as well as cells and other plantparts thereof. Such plants may be developed using the markers identifiedin WO 00/31964, U.S. Pat. No. 6,162,967 and U.S. application Ser. No.09/954,773. Progeny plants and plant parts produced using hybrid tomato‘E2134649’ may be identified by having a molecular marker profile of atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% genetic contributionfrom hybrid tomato ‘E2134649’, as measured by either percent identity orpercent similarity. Such progeny may be further characterized as beingwithin a pedigree distance of hybrid tomato ‘E2134649’, such as within1, 2, 3, 4 or 5 or less cross-pollinations to a tomato plant other thanhybrid tomato ‘E2134649’ or a plant that has hybrid tomato ‘E2134649’ asa progenitor. Unique molecular profiles may be identified with othermolecular tools such as SNPs and RFLPs.

While determining the SSR genetic marker profile of the plants describedsupra, several unique SSR profiles may also be identified which did notappear in either parent of such plant. Such unique SSR profiles mayarise during the breeding process from recombination or mutation. Acombination of several unique alleles provides a means of identifying aplant variety, an F₁ progeny produced from such variety, and progenyproduced from such variety.

Single-Gene Conversions

When the term “tomato plant” is used in the context of the presentdisclosure, this also includes any single gene conversions of thatvariety. The term single gene converted plant as used herein refers tothose tomato plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique. Backcrossing methods can be used with the present disclosureto improve or introduce a characteristic into the variety. The term“backcrossing” as used herein refers to the repeated crossing of ahybrid progeny back to the recurrent parent, i.e., backcrossing 1, 2, 3,4, 5, 6, 7, 8 or more times to the recurrent parent. The parental tomatoplant that contributes the gene for the desired characteristic is termedthe nonrecurrent or donor parent. This terminology refers to the factthat the nonrecurrent parent is used one time in the backcross protocoland therefore does not recur. The parental tomato plant to which thegene or genes from the nonrecurrent parent are transferred is known asthe recurrent parent as it is used for several rounds in thebackcrossing protocol (Poehlman & Sleper, 1994; Fehr, Principles ofCultivar Development pp. 261-286 (1987)). In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the single geneof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a tomato plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add an agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic; examples of these traits include, for example, malesterility, herbicide resistance, resistance for bacterial, fungal, orviral disease, insect resistance, male fertility, enhanced nutritionalquality, industrial usage, yield stability and yield enhancement. Thesegenes are generally inherited through the nucleus. Several of thesesingle gene traits are described in U.S. Pat. Nos. 5,959,185; 5,973,234and 5,977,445.

Tissue Culture

Further reproduction of a tomato variety can occur by tissue culture andregeneration. Tissue culture of various tissues of tomatoes andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Girish-Chandel et al., Advances inPlant Sciences, 2000, 13: 1, 11-17; Costa et al., Plant Cell Report,2000, 19: 3 327-332; Plastira et al., Acta Horticulturae, 1997, 447,231-234; Zagorska et al., Plant Cell Report, 1998, 17: 12 968-973;Asahura et al., Breeding Science, 1995, 45: 455-459; Chen et al.,Breeding Science, 1994, 44: 3, 257-262, Patil et al., Plant and Tissueand Organ Culture, 1994, 36: 2, 255-258; Gill, R., et al., SomaticEmbryogenesis and Plant Regeneration from Seedling Cultures of Tomato(Lycopersicon esculentum Mill.), J. Plant Physiol., 147:273-276 (1995);Jose M. Segui-Simarro and Fernando Nuez, Embryogenesis induction,callogenesis, and plant regeneration by in vitro culture of tomatoisolated microspores and whole anthers J. Exp. Bot., March 2007; 58:1119-1132; Hamza et al., Re-evaluation of Conditions for PlantRegeneration and Agrobacterium-Mediated Transformation from Tomato(Lycopersicon esculentum), J. Exp. Bot., December 1993; 44: 1837-1845.Thus, another aspect of this disclosure is to provide cells which upongrowth and differentiation produce tomato plants having thephysiological and morphological characteristics of hybrid tomato‘E2134649’.

As used herein, the term “tissue culture” indicates a compositioncontaining isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, fruit,petioles, leaves, stems, roots, root tips, anthers, pistils and thelike. Means for preparing and maintaining plant tissue culture are wellknown in the art. By way of example, a tissue culture containing organshas been used to produce regenerated plants. U.S. Pat. Nos. 5,959,185;5,973,234 and 5,977,445 describe certain techniques.

Vegetative Propagation

Tomato plants can also be propagated vegetatively. Accordingly, thepresent disclosure is further directed to vegetative propagation ofhybrid tomato ‘E2134649’. A part of the plant, for example a shoottissue, is collected and a new plant is obtained from the part. Suchpart typically includes an apical meristem of the plant. The collectedpart is transferred to a medium allowing development of a plantletincluding, for example, rooting or development of shoots, or is graftedonto a tomato plant or a rootstock prepared to support growth of shoottissue. This is achieved using methods well-known in the art.Accordingly, in one embodiment, a method of vegetatively propagating atomato plant of the present disclosure involves collecting a part of aplant according to the present disclosure, e.g. a shoot tissue, andobtaining a plantlet from said part. In one embodiment, a method ofvegetatively propagating a tomato plant of the present disclosureinvolves: a) collecting tissue of a plant of the present disclosure; andb) rooting said proliferated shoots to obtain rooted plantlets. In oneembodiment, a method of vegetatively propagating a plant of the presentdisclosure involves: a) collecting tissue of a plant of the presentdisclosure; b) cultivating said tissue to obtain proliferated shoots;and c) rooting said proliferated shoots to obtain rooted plantlets. Inone embodiment, such methods further involve growing a plant from saidplantlets. In one embodiment, a fruit is harvested from said plant.

Additional Breeding Methods

Tomato varieties such as hybrid tomato ‘E2134649’ are typicallydeveloped for use as fresh produce or for processing. However, tomatovarieties also provide a source of breeding material that may be used todevelop new tomato varieties. Plant breeding techniques known in the artand used in a tomato plant breeding program may include, for example,chasing selfs, recurrent selection, mass selection, bulk selection,mutation breeding, backcrossing, pedigree breeding, open pollinationbreeding, restriction fragment length polymorphism enhanced selection,genetic marker enhanced selection, making double haploids, andtransformation. Often combinations of these techniques are used. Thedevelopment of tomato varieties in a plant breeding program involves, ingeneral, the development and evaluation of homozygous varieties. Thereare many analytical methods available to evaluate a new variety. Theoldest and most traditional method of analysis is the observation ofphenotypic traits but genotypic analysis may also be used. Thus, anotheraspect of the disclosure is to provide hybrid tomato ‘E2134649’ as asource of breeding material for the development of new tomato varietiesusing, for example, the breeding techniques described herein. One ofskill in the art would recognize that additional breeding techniques mayexist and may be used to develop new tomato varieties using hybridtomato ‘E2134649’.

The present disclosure is directed to methods for producing a tomatoplant by crossing a first parent tomato plant with a second parenttomato plant where either the first or second parent tomato plant ishybrid tomato ‘E2134649’. The other parent may be any other tomatoplant, such as a tomato plant that is part of a synthetic or naturalpopulation. Any such methods using hybrid tomato ‘E2134649’ are part ofthis disclosure: selfing, sibbing, backcrosses, mass selection, pedigreebreeding, bulk selection, hybrid production, crosses to populations, andthe like. These methods are well known in the art and some of the morecommonly used breeding methods are described herein. Descriptions ofbreeding methods can be found in one of several reference books (e.g.,Allard, Principles of Plant Breeding, 1960; Simmonds, Principles of CropImprovement, 1979; Sneep et al., 1979; Fehr, “Breeding Methods forCultivar Development,” 2.sup.nd ed., Wilcox editor, 1987).

The following describes breeding methods that may be used with hybridtomato ‘E2134649’ in the development of further tomato plants. One suchembodiment is a method for developing a ‘E2134649’ progeny tomato plantin a tomato plant breeding program involving: obtaining the tomatoplant, or a part thereof, of ‘E2134649’, utilizing said plant or plantpart as a source of breeding material, and selecting an ‘E2134649’progeny plant with molecular markers in common with ‘E2134649’ and/orwith morphological and/or physiological characteristics selected fromthe characteristics listed in Table 1. Breeding steps that may be usedin the tomato plant breeding programs may include pedigree breeding,backcrossing, mutation breeding, and recurrent selection. In conjunctionwith these steps, techniques such as RFLP-enhanced selection, geneticmarker enhanced selection (for example SSR markers) and the making ofdouble haploids may be utilized.

Another method involves producing a population of ‘E2134649’ progenytomato plants, involving crossing hybrid tomato ‘E2134649’ with anothertomato plant, thereby producing a population of tomato plants, which, onaverage, derive 50% of their alleles from ‘E2134649’. A plant of thispopulation may be selected and repeatedly selfed or sibbed with a tomatocultivar resulting from these successive filial generations. In oneembodiment, the tomato cultivar produced by this method has obtained atleast 50% of its alleles from ‘E2134649’.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus, the disclosure includes ‘E2134649’progeny tomato plants containing a combination of at least two traits ofhybrid tomato ‘E2134649’, the traits being selected from those listed inTables 1 and 2, so that the progeny tomato plant is not significantlydifferent for the traits than ‘E2134649’ as determined at the 5%significance level when grown in the same environmental conditions.Using techniques described herein, molecular markers may be used toidentify said progeny plant as a ‘E2134649’ progeny plant. For each ofthe evaluation schemes involving hybrid tomato ‘E2134649’, mean traitvalues may be used to determine whether trait differences aresignificant, and preferably the traits are measured on plants grownunder the same environmental conditions. Once such a variety isdeveloped its value is substantial since it is important to advance thegermplasm base as a whole in order to maintain or improve traits such asyield, disease resistance, pest resistance, and plant performance inextreme environmental conditions.

Progeny of ‘E2134649’ may also be characterized through their filialrelationship with ‘E2134649’, as for example being within a certainnumber of breeding crosses ‘E2134649’. A breeding cross is a cross madeto introduce new genetics into the progeny, and is distinguished from across, such as a self or a sib cross, made to select among existinggenetic alleles. The lower the number of breeding crosses in thepedigree, the closer the relationship between ‘E2134649’ and itsprogeny. For example, progeny produced by the methods described hereinmay be within 1, 2, 3, 4 or 5 breeding crosses of ‘E2134649’.

Exemplary breeding techniques are further described herein and may beused in breeding schemes using hybrid tomato ‘E2134649’.

Chasing Selfs

Chasing selfs involves identifying inbred plants among tomato plantsthat have been grown from hybrid tomato seed, such as the seed fromhybrid tomato ‘E2134649’. Once the seed is planted, the inbred plantsmay be identified and selected due to their decreased vigor relative tothe hybrid plants that grow from the hybrid seed. By locating the inbredplants, isolating them from the rest of the plants, and self-pollinatingthem (i.e., “chasing selfs”), a breeder can obtain an inbred line thatis identical to an inbred parent used to produce the hybrid.

Accordingly, another aspect of the present disclosure relates to amethod for producing an inbred tomato variety by: planting seed of thehybrid tomato ‘E2134649’; growing plants from the seed; identifying oneor more inbred tomato plants; controlling pollination in a manner whichpreserves homozygosity of the one or more inbred plants; and harvestingresultant seed from the one or more inbred plants. The step ofidentifying the one or more inbred tomato plants may further includeidentifying plants with decreased vigor, i.e., plants that appear lessrobust than plants of the hybrid tomato ‘E2134649’. Tomato plantscapable of expressing essentially all of the physiological andmorphological characteristics of the parental inbred lines of hybridtomato ‘E2134649’ include tomato plants obtained by chasing selfs fromseed of hybrid tomato ‘E2134649’.

One of ordinary skill in the art will recognize that once a breeder hasobtained inbred tomato plants by chasing selfs from seed of hybridtomato ‘E2134649’, the breeder can then produce new inbred plants suchas by sib-pollinating, or by crossing one of the identified inbredtomato plant with a plant of the hybrid tomato ‘E2134649’.

Backcross Conversion

Hybrid tomato ‘E2134649’ represents a new base genetic variety intowhich a new locus or trait may be introgressed. Backcrossing representsan important method that can be used to accomplish such anintrogression. The term backcross conversion and single locus conversionare used interchangeably to designate the product of a backcrossingprogram.

A backcross conversion of a tomato variety such as, for example, hybridtomato ‘E2134649’, occurs when DNA sequences are introduced throughbackcrossing (Hallauer et al, 1988, “Corn Breeding” Corn and CornImprovements, No. 18, pp. 463-481), with the tomato variety utilized asthe recurrent parent. Both naturally occurring and transgenic DNAsequences may be introduced through backcrossing techniques. A backcrossconversion may produce a plant with a trait or locus conversion in atleast two or more backcrosses, including at least 2 crosses, at least 3crosses, at least 4 crosses, at least 5 crosses and the like. Molecularmarker assisted breeding or selection may be utilized to reduce thenumber of backcrosses necessary to achieve the backcross conversion. Forexample, see Openshaw, S. J. et al., Marker-assisted Selection inBackcross Breeding. In: Proceedings Symposium of the Analysis ofMolecular Data, August 1994, Crop Science Society of America, Corvallis,Oreg., where it is demonstrated that a backcross conversion can be madein as few as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes as vs.unlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear) and the types of parents includedin the cross. Desired traits that may be transferred through backcrossconversion may include, for example, sterility (nuclear andcytoplasmic), fertility restoration, nutritional enhancements, droughttolerance, nitrogen utilization, industrial enhancements, diseaseresistance (bacterial, fungal or viral), insect resistance and herbicideresistance. In addition, an introgression site itself, such as an FRTsite, Lox site or other site specific integration site, may be insertedby backcrossing and utilized for direct insertion of one or more genesof interest into a specific plant variety. In some embodiments of thedisclosure, the number of loci that may be backcrossed into a tomatovariety such as, for example, hybrid tomato ‘E2134649’, is at least 1,2, 3, 4, or 5 and/or no more than 6, 5, 4, 3, or 2. A single locus maycontain several transgenes, such as a transgene for disease resistancethat, in the same expression vector, also contains a transgene forherbicide resistance. The gene for herbicide resistance may be used as aselectable marker and/or as a phenotypic trait. A single locusconversion of site specific integration system allows for theintegration of multiple genes at the converted loci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele involves growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may involve additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. The backcross is a form ofinbreeding, and the features of the recurrent parent are automaticallyrecovered after successive backcrosses. Poehlman, Breeding Field Crops,P. 204 (1987). Poehlman suggests from one to four or more backcrosses,but as noted above, the number of backcrosses necessary can be reducedwith the use of molecular markers. Other factors, such as a geneticallysimilar donor parent, may also reduce the number of backcrossesnecessary. As noted by Poehlman, backcrossing is easiest for simplyinherited, dominant and easily recognized traits.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such as‘E2134649’ and another tomato variety having one or more desirablecharacteristics that is lacking or which complements ‘E2134649’. If thetwo original parents do not provide all the desired characteristics,other sources can be included in the breeding population. In thepedigree method, superior plants are selfed and selected in successivefilial generations. In the succeeding filial generations theheterozygous condition gives way to homogeneous varieties as a result ofself-pollination and selection. Typically in the pedigree method ofbreeding, five or more successive filial generations of selfing andselection is practiced: F₁ to F₂; F₂ to F₃; F₃ to F₄; F₄ to F₅, etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed variety. Preferably, thedeveloped variety contains homozygous alleles at about 95% or more ofits loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodcharacteristics yet lacks that desirable trait or traits. However, thesame procedure can be used to move the progeny toward the genotype ofthe recurrent parent but at the same time retain many components of thenon-recurrent parent by stopping the backcrossing at an early stage andproceeding with selfing and selection. For example, a tomato variety maybe crossed with another variety to produce a first generation progenyplant. The first generation progeny plant may then be backcrossed to oneof its parent varieties to create a BC1 or BC2. Progeny are selfed andselected so that the newly developed variety has many of the attributesof the recurrent parent and yet several of the desired attributes of thenon-recurrent parent. This approach leverages the value and strengths ofthe recurrent parent for use in new tomato varieties.

Therefore, an embodiment of this disclosure is a method of making abackcross conversion of ‘E2134649’, involving the steps of crossing aplant of ‘E2134649’ with a donor plant having a desired trait, selectingan F₁ progeny plant having the desired trait, and backcrossing theselected F₁ progeny plant to a plant of ‘E2134649’. This method mayfurther involve the step of obtaining a molecular marker profile of‘E2134649’ and using the molecular marker profile to select for aprogeny plant with the desired trait and the molecular marker profile of‘E2134649’. In one embodiment the desired trait is a mutant gene ortransgene present in the donor parent.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. The method entails individual plantscross pollinating with each other to form progeny. The progeny are grownand the superior progeny selected by any number of selection methods,which include individual plant, half-sib progeny, full-sib progeny andselfed progeny. The selected progeny are cross pollinated with eachother to form progeny for another population. This population is plantedand again superior plants are selected to cross pollinate with eachother. Recurrent selection is a cyclical process and therefore can berepeated as many times as desired. The objective of recurrent selectionis to improve the traits of a population. The improved population canthen be used as a source of breeding material to obtain new varietiesfor commercial or breeding use, including the production of a syntheticcultivar. A synthetic cultivar is the resultant progeny formed by theintercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection, seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection may involve growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self-pollination, directed pollinationcould be used as part of the breeding program.

Thus, another aspect of the disclosure is the use of ‘E2134649’ inrecurrent selection and/or mass selection breeding schemes and may beused to develop new tomato varieties.

Mutation Breeding

Mutation breeding is another method of introducing new traits intohybrid tomato ‘E2134649’. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including, for example, temperature, long-termseed storage, tissue culture conditions, radiation; such as X-rays,Gamma rays (e.g. cobalt 60 or cesium 137), neutrons, (product of nuclearfission by uranium 235 in an atomic reactor), Beta radiation (emittedfrom radioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in“Principles of Cultivar Development” Fehr, 1993 Macmillan PublishingCompany. In addition, mutations created in other tomato plants may beused to produce a backcross conversion of hybrid tomato ‘E2134649’ thatincludes such mutation.

Breeding with Molecular Markers

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs) and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing hybrid tomato ‘E2134649’.

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. See, for example, Shoemaker and Olsen, ((1993)Molecular Linkage Map of Soybean (Glycine max L. Men.). p. 6.131-6.138.In S. J. O'Brien (ed.) Genetic Maps: Locus Maps of Complex Genomes. ColdSpring Harbor Laboratory Press. Cold Spring Harbor, N.Y.), developed amolecular genetic linkage map that consisted of 25 linkage groups withabout 365 RFLP, 11 RAPD (random amplified polymorphic DNA), threeclassical markers, and four isozyme loci. See also, Shoemaker R. C. 1994RFLP Map of Soybean. P. 299-309 In R. L. Phillips and I. K. Vasil (ed.)DNA-based markers in plants. Kluwer Academic Press Dordrecht, theNetherlands.

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. For exampleDiwan and Cregan, described a highly polymorphic microsatellite loci intomato with as many as 26 alleles. (Diwan, N., and P. B. Cregan 1997Automated sizing of fluorescent-labeled simple sequence repeat (SSR)markers to assay genetic variation in Soybean Theor. Appl. Genet.95:220-225.) Single Nucleotide Polymorphisms may also be used toidentify the unique genetic composition of the tomato plants describedherein and progeny varieties retaining that unique genetic composition.Various molecular marker techniques may be used in combination toenhance overall resolution.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Production of Double Haploids

The production of double haploids may also be used for the developmentof plants with a homozygous genotype and/or phenotype in the breedingprogram. For example, a tomato plant for which ‘E2134649’ is a parentcan be used to produce double haploid plants. Double haploids areproduced by the doubling of a set of chromosomes (1 N) from aheterozygous plant to produce a completely homozygous individual. Forexample, see Wan et al., “Efficient Production of Doubled Haploid PlantsThrough Colchicine Treatment of Anther-Derived Maize Callus”,Theoretical and Applied Genetic, 77:889-892, 1989 and U.S. Pat. No.7,135,615. This can be advantageous because the process omits thegenerations of selfing needed to obtain a homozygous plant from aheterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selected line(as female) with an inducer line. Such inducer lines for maize includeStock 6 (Coe, 1959, Am. Nat. 93:381-382; Sharkar and Coe, 1966, Genetics54:453-464), KEMS (Deimling, Roeber, and Geiger, 1997, Vortr.Pflanzenzuchtg 38:203-224), or KMS and ZMS (Chalyk, Bylich & Chebotar,1994, MNL 68:47; Chalyk & Chebotar, 2000, Plant Breeding 119:363-364),and indeterminate gametophyte (ig) mutation (Kermicle 1969 Science166:1422-1424).

Methods for obtaining haploid plants are also disclosed in Kobayashi, M.et al., Journ. Heredity 71(1):9-14, 1980, Pollacsek, M., Agronomie(Paris) 12(3):247-251, 1992; Cho-Un-Haing et al., Journ. of Plant Biol.,1996, 39(3):185-188; Verdoodt, L., et al., February 1998, 96(2):294-300;Genetic Manipulation in Plant Breeding, Proceedings InternationalSymposium Organized by EUCARPIA, Sep. 8-13, 1985, Berlin, Germany;Chalyk et al., 1994, Maize Genet Coop. Newsletter 68:47; Chalyk, S.

Thus, an embodiment of this disclosure is a process for making asubstantially homozygous ‘E2134649’ progeny plant by producing orobtaining a seed from the cross of ‘E2134649’ and another tomato plantand applying double haploid methods to the F₁ seed or F₁ plant or to anysuccessive filial generation. Based on studies in maize and currentlybeing conducted in tomato, such methods would decrease the number ofgenerations required to produce a variety with similar genetics orcharacteristics to ‘E2134649’. See Bernardo, R. and Kahler, A. L.,Theor. Appl. Genet. 102:986-992, 2001.

In particular, a process of making seed retaining the molecular markerprofile of ‘E2134649’ is contemplated, such process involving obtainingor producing F₁ seed for which ‘E2134649’ is a parent, inducing doubledhaploids to create progeny without the occurrence of meioticsegregation, obtaining the molecular marker profile of ‘E2134649’, andselecting progeny that retain the molecular marker profile of‘E2134649’.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the disclosure (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments of the disclosure and does not pose alimitation on the scope of the disclosure unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the embodiments ofthe disclosure.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

Deposit Information

A deposit of the hybrid tomato ‘E2134649’ is maintained by Enza ZadenUSA, Inc., having an address at 7 Harris Place, Salinas, Calif. 93901,United States. Access to this deposit will be available during thependency of this application to persons determined by the Commissionerof Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14and 35 U.S.C. §122. Upon allowance of any claims in this application,all restrictions on the availability to the public of the variety willbe irrevocably removed by affording access to a deposit of at least2,500 seeds of the same variety with the American Type CultureCollection, (ATCC), P.O. Box 1549, MANASSAS, VA 20108 USA.

Applicants have made available to the public without restriction adeposit of at least 2500 seeds of hybrid tomato ‘E2134649’ with theAmerican Type Culture Collection (ATCC), P.O. Box 1549, MANASSAS, VA20108 USA, with a deposit on (DATE) which has been assigned ATCC numberX1.

The deposit will be maintained in the ATCC depository, which is a publicdepository, for a period of 30 years, or 5 years after the most recentrequest, or for the effective life of the patent, whichever is longer,and will be replaced if a deposit becomes nonviable during that period.

What is claimed is:
 1. A hybrid tomato seed designated as ‘E2134649’having ATCC Accession Number X1.
 2. A tomato plant produced by growingthe seed of claim
 1. 3. A plant part from the plant of claim
 2. 4. Theplant part of claim 3, wherein said part is a leaf, an ovule, pollen, aseed, a fruit, a cell, or a portion thereof.
 5. A tomato plant havingall the physiological and morphological characteristics of the tomatoplant of claim
 2. 6. A plant part from the plant of claim
 5. 7. Theplant part of claim 6, wherein said part is a leaf, an ovule, pollen, aseed, a fruit, a cell, or a portion thereof.
 8. An F₁ hybrid tomatoplant having ‘E2134649’ as a parent where ‘E2134649’ is grown from theseed of claim
 1. 9. Pollen or an ovule of the plant of claim
 2. 10. Aprotoplast produced from the plant of claim
 2. 11. A tissue culture ofthe plant of claim
 2. 12. The tissue culture of claim 11, wherein saidtissue culture is produced from a plant part selected from the groupconsisting of leaf, anther, pistil, stem, petiole, root, root tip,fruit, seed, flower, cotyledon, hypocotyl, embryo and meristematic cell.13. A tomato plant regenerated from the tissue culture of claim 11,wherein the plant has all of the morphological and physiologicalcharacteristics of a tomato plant produced by growing seed designated as‘E2134649’ having ATCC Accession Number X1.
 14. A method of makingtomato seeds, said method comprising crossing the plant of claim 2 withanother tomato plant and harvesting seed therefrom.
 15. A method ofmaking hybrid tomato ‘E2134649’, said method comprising selecting seedsfrom the cross of one ‘E2134649’ plant with another ‘E2134649’ plant, asample of ‘E2134649’ tomato seed having been deposited under ATCCAccession Number X1.